The present invention relates to a method and apparatus for analyzing a blood or other biological fluid sample in a quiescent state, whereby particulate constituents of biological samples that contain sparse populations of cellular species of interest can be enumerated and inspected using an optical scanning instrument. Specifically, this invention relates to a method and apparatus for obtaining an increased cellular or particulate concentration within the use of said optical scanning method.
The formation of appropriate cellular or particulate layers for later optical examination is important to many fields. One of these fields is hematology, where several methods and devices have been described for obtaining clinically useful cell concentrations.
A method and apparatus for analyzing a blood or other biologic fluid sample in a quiescent state without the need for separate fluid streams passing through the blood sample during the analysis is described in U.S. Ser. No. 09/248,135 filed Feb. 10, 1999, now U.S. Pat. No. 6,123,184, and U.S. Ser. No. 09/249,721 filed Feb. 12, 1999, now U.S. Pat. No. 6,235,536. Although this method simplifies the analysis procedure and yields the full compliment of CBC parameters it also possesses several disadvantages. One disadvantage of the apparatus is that the concentration of cells in the examination layer is not controlled. This can lead to difficulties in optically examining cell volume and morphology. Another disadvantage of the aforementioned apparatus is that the field of cells may be too sparse in clinically relevant samples to complete scanning in a timely manner.
U.S. Pat. Nos. 5,627,041 and 5,912,134 describe an apparatus and method for cytometric measurement of cell populations using fluorescent markers. However, a disadvantage of this method is that, if the sample under test is blood, it requires addition of diluent in such quantities that white blood cells (WBCs) with depressed counts are not very numerous in the sample and may require extremely long examination times to locate them. Moreover, if the cell counts within the undiluted blood are low, clinically relevant cell populations present in the sample may not be detected in the diluted sample. For example, patients who undergo chemotherapy regimens may have depressed white cell counts in the range of 1000 cells/xcexcL and less. Cytometric examinations are typically searching for a sub-population of these cells, further reducing the likelihood of locating them.
U.S. Pat. No. 4,790,640 discloses a wedge shaped device for trapping rigid particles, such as sickle cells in blood. However, a disadvantage of the device is that the selection of cell sizes is accomplished by thickness of the chamber alone, which can exhibit substantial manufacturing variation over the examination area, causing a corresponding loss of ability to separate by size.
Consequently, it would be desirable to have a method and apparatus for obtaining the desired cellular concentration in a blood or other biologic sample which can mitigate the effect of a separate dilution step and addition of diluting fluids.
It is an objective of the present invention to provide channels in a separation wall inside a separation chamber, said channels having appropriate size and dimensions to allow at least one undesired particle species to pass while excluding larger particles from passing through, thereby arriving at a predetermined increased volume fraction of the desired particles.
It is another objective of the present invention to incorporate at least two separating channels in a separation wall in a separation chamber, the channels having channel sizes selected to allow at least one cell species and the substantially liquid component of the sample to pass through them, arriving at a desired concentration of larger cell types in the first compartment in front of the separation wall. A further embodiment of the present invention is to have a plurality of separating channels in the separating wall having one channel size selected to allow at least one cell species and the substantially liquid component of the sample to pass through them.
It is a further objective of the present invention to regulate the volume fraction of cellular or particle components of a specimen by means of an array of separating channels which effect their selection by means of size exclusion during flow between two adjoining compartments.
It is another objective of the present invention to increase the concentration of larger particles for cytometric examination of sparse populations by allowing only smaller particles and substantially liquid components to pass through the channels into a subsequent chamber.
It is a still further objective of the present invention to create an accurate spacing between two opposing containment walls to allow for the optimal formation of desired regions where all particles of interest lie in the same focal plane, allowing an accurate determination of the chamber thickness without relying on extraneous equipment and manipulations for height calibration.